Double-target chimeric antigen receptor targeting CLL1 and NKG2D ligands and application thereof

A chimeric antigen receptor and dual-target technology, applied in the field of biomedicine, can solve problems such as reducing the recurrence rate of the disease, and achieve the effects of reducing the probability of disease recurrence, solving the problem of tumor antigen heterogeneity, and achieving a significant killing effect.

Active Publication Date: 2021-08-13
GUANGZHOU BIO GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, there are currently few CAR-T technologies that co-ta

Method used

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  • Double-target chimeric antigen receptor targeting CLL1 and NKG2D ligands and application thereof
  • Double-target chimeric antigen receptor targeting CLL1 and NKG2D ligands and application thereof
  • Double-target chimeric antigen receptor targeting CLL1 and NKG2D ligands and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0096] Example 1 Design of chimeric antigen receptor

[0097] In this embodiment, anti-CL1 antibody H27H4 (SEQ ID NO: 1 to 2), 1075.7 (SEQ ID NO: 3 to 4) or 27H4 (SEQ ID NO: 5 ~ 6) and NKG2D extracellular segment (NKG2D-ECD, SEQ) ID NO: 7) As an antigen binding domain, bind to CD8α signal peptide (SEQ ID NO: 16 to 17) or IgGκ light chain signal peptide (SEQ ID NO: 34 ~ 35), CD8α hinge region (SEQ ID NO: 18 ~ 19) and transmembrane regions (SEQ ID NO: 20 ~ 21), 4-1BB co-stimulation domain (SEQ ID NO: 22 ~ 23) and CD3 ζ signal conduction domains (SEQ ID NO: 24 ~ 25), constructing a target Double target Car (SEQ ID NO: 8 ~ 13), structure diagram such as CLL1 and NKG2D ligand (NKG2DL), structure Figure 1A Indicated;

[0098] This embodiment also constructs anti-CL1 single target CAR (SEQ ID NO: 26 ~ 27, SEQ ID NO: 28 ~ 29, SEQ ID NO: 30 ~ 31) and anti-NKG2DL single target CAR (SEQ ID NO: 32 ~ 33), structure diagram Figure 1B Indicated.

[0099] Information about Car molecules is shown ...

Example Embodiment

[0102] Example 2 Construction of chimeric antigen receptor expression vector

[0103] (1) CAR molecules designated in Example 1, codon optimization of CAR coding genes, promoting its efficient expression in human cells, all gene synthesis CAR coding gene (Guangzhou Aiki Biotechnology Co., Ltd.);

[0104] (2) After 30 minutes of EcoRI and BamHi bisase cutting length CAR gene and empty carrier PCDH-EF1-MCS, 1.5% agarose gel was used for DNA electrophoresis using 1.5% agarose gel and purified using agarose gel. The recovery kit (mean) purifies the enzyme digestion;

[0105] (3) Preparation of the connection system shown in Table 2, 22 ° C for the Car gene fragment and the linearized PCDH-EF1-MCS, directly transform the connection product directly to the STBL3 Escherichia coli, take 200 μL of transformed product to coating ampican resistance Sexual LB tablet, inverted in the 37 ° C incubator overnight, three monoclons were randomly selected for colonial PCR identification, and sequenc...

Example Embodiment

[0109] Example 3 Slow Virus Packaging

[0110] This example uses a quadruplicate system to perform a slow viral package for a slow viral expression vector, and the specific steps are as follows:

[0111] (1) After mixing a lentiviral expression vector, the auxiliary plasmid GAG / POL, REV, and VSV-G, after mixing with the PEI transfection reagent, adding to a certain volume of serum DMEM, mixing for 15 min;

[0112] (2) The above mixture is added to the T75 cell culture flask with 293T cells, gently mixed, at 37 ° C, 5% CO 2 Culture in cell incubator for 6 h;

[0113] (3) After 6 hours, fresh medium was replaced, continued to cultivate, and sodium phthal sodium butyrate; 72 h collection of chronic viral culture supernatant was purified.

[0114] Packaging lentiviral titers are shown in Table 3.

[0115] table 3

[0116] CAR slow virus Tool (TU / mL) NKG2D-Car 8.35E7 H27H4-Car 1.56E8 H27H4-NKG2D-Car 1.48E8 NKG2D-H27H4-Car 1.52E8 1075.7-Car 5.67E7 ...

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Abstract

The invention provides a double-target chimeric antigen receptor targeting CLL1 and a NKG2D ligand and application thereof. The double-target chimeric antigen receptor comprises a signal peptide, an antigen binding structural domain, a hinge region, a transmembrane structural domain and a signal transduction structural domain, and the antigen binding domain comprises an anti-CLL1 antibody and NKG2D. The CAR molecule simultaneously targeting CLL1 and the NKG2D ligand is constructed, the effect of comprehensively targeting acute myelogenous leukemia cells is achieved, the CAR-T cells for expressing the double-target CAR targeting CLL1 and the NKG2D ligand are remarkable in tumor removal effect, the antigen escape phenomenon is avoided, and the CAR molecule has important significance in the field of tumor treatment.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and relates to a dual-target chimeric antigen receptor targeting CLL1 and NKG2D ligands and an application thereof. Background technique [0002] In the field of tumor immunotherapy, CAR-T is the most popular tumor immunotherapy method, and it has shown amazing effects in the treatment of leukemia, lymphoma, and multiple myeloma. At present, research on CAR-T products targeting CD19 is relatively in-depth. Kymriah and Yescarta approved in the United States are both CAR-T products targeting CD19 for the treatment of hematological tumors. From a global perspective, the R&D pipeline of CAR-T is rapidly expanding, including the exploration of new targets, such as BCMA, CD123, CD33, etc., and the expansion of new indications, such as the advancement from hematological tumors to solid tumors. The projects of many companies around the world have advanced to the clinical stage, and it is expected th...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K16/2851C07K14/7056C07K14/7051C07K14/70517C07K14/70578C12N15/86C12N5/0636A61K39/001102A61K39/001122A61P35/00C07K2317/56C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/15043C12N2800/107C12N2510/00A61K2039/5156A61K2039/804
Inventor 李光超罗敏周兆丁雯王学俊
Owner GUANGZHOU BIO GENE TECH CO LTD
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